To test the expression of H2S-generating enzymes, including CBS, CSE and 3-MPST, in the human thyroid, the thyroid tissue sections mentioned above were microwaved in citrate buffer or EDTA buffer for 15 min, and then 3% hydrogen peroxide was used to block endogenous peroxidase. After blocking with 3% bovine serum albumin (BSA, Sigma–Aldrich), tissue slides were incubated with the following primary antibodies at 4°C overnight: CBS antibody (1:200), CSE antibody (1:25), and 3-MPST antibody (1:25) (all from Santa Cruz, California, United States). Then, the slides were incubated with secondary antibody for 60 min. Finally, 3,3′-diaminobenzidine (DAB) staining (ZSGB–BIO, Beijing, China) was used to detect positive areas, which were monitored by an Olympus BX51T microscope (Tokyo, Japan).
3 3 diaminobenzidine dab staining
Manufactured by ZSGB-BIO
Sourced in China
3,3'-diaminobenzidine (DAB) is a chromogenic substrate used in immunohistochemistry and in situ hybridization techniques. It produces a brown precipitate at the site of an antigen-antibody or nucleic acid-probe reaction, allowing for the visualization and localization of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Bx51t microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cbs antibody, by Santa Cruz Biotechnology (1 mentions)
Cse antibody, by Santa Cruz Biotechnology (1 mentions)
Anti collagen 1 antibody, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Optical microscope, by Nikon (1 mentions)
Anti collagen 2 antibody, by Abcam (1 mentions)
Caseviewer, by 3DHISTECH (1 mentions)
Tmprss2, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Anti ly6g, by Abcam (1 mentions)
Anti cd68, by Immunoway (1 mentions)
Anti nlrp3, by Boster Bio (1 mentions)
Fabp4, by Proteintech (1 mentions)
Digitalized microscope camera, by Olympus (1 mentions)
6 protocols using 3 3 diaminobenzidine dab staining
1
Thyroid H2S-generating Enzyme Expression
2
Immunohistochemical Analysis of Cardiac Collagen
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The paraffin sections of the left ventricular myocardial tissue were prepared as described above. After antigen retrieval with citrate buffer, 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. Sections were blocked with 0.05% bovine serum albumin (BSA; GIBCO, CA, USA) for 1 hour at room temperature, followed by incubation with primary antibodies (1:400 anti-collagen I antibody and 1:400 anti-collagen II antibody; Abcam, CA, USA) at 4 °C overnight. Sections were then incubated for 1 hour with a horseradish peroxidase (HRP)-labeled secondary antibody, followed by 3,3'-diaminobenzidine (DAB) staining (ZSbio, Beijing, China). A positive DAB stain can be visualized as a specific brown-yellow color. Finally, sections were dehydrated using an ethanol gradient, treated with xylene and sealed. Slides were observed under an optical microscope (Nikon, Tokyo, Japan) with 400× magnification.
3
TRAF6 Immunohistochemistry Protocol
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After washing with PBS, tissues were fixed in 10% formaldehyde for 4–6 h at RT, followed by routine deparaffinization and dehydration for Immunohistochemistry (IHC). Formalin‐fixed paraffin‐embedded tissues were cut into 4 μm sections. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide and nonspecific binding with 5% bovine serum albumin for 20 min. Tissue sections were incubated with primary antibodies targeting TRAF6 for 1 h at 37°C and then washed three times with PBS plus 1% Tween‐20 (PBST) for 3 min. Antibody binding was detected with a brown precipitate followed by 3,3‐diaminobenzidine (DAB) staining (ZSGB BIO, China), haematoxylin counterstaining and dehydration. IHC slides were scanned by a Pannoramic DESK scanner and analysed by CaseViewer (3DHISTECH, Hungary).
4
Immunohistochemical Analysis of COVID-19 Receptors
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Human thyroid tissue slides were antigen repaired in EDTA buffer for 20 min, and endogenous peroxidase was then blocked with 3% hydrogen peroxide. After blocking with 3% bovine serum albumin (BSA, Sigma–Aldrich, St. Louis, MO, USA), the slides were incubated with the following antibodies overnight: ACE2 (1:500; Proteintech, Wuhan, China), NRP1 (1:500; Proteintech, Wuhan, China), and TMPRSS2 (1:1000, Abcam, Cambridge, UK). Afterward, the slides were incubated with the respective secondary antibodies for 60 min. Finally, positive areas were detected by 3,3′-diaminobenzidine (DAB) staining (ZSGB–BIO, Beijing, China) and observed with an Olympus BX51T microscope (Tokyo, Japan). Immunostaining intensities were assessed by histological scores (H-scores) as described previously [61 (link)].
5
Quantifying Immune Cell Profiles in Lung Tissue
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Lung tissue sections were thoroughly deparaffinized, rehydrated, and placed in citrate buffer (pH=6) for antigen retrieval. Then, the sections were incubated in 3% hydrogen peroxide buffer to eliminate endogenous peroxidase. The tissues were blocked at room temperature and subsequent incubation with rat polyclonal anti-LY6G (1:150, Abcam, UK), mouse polyclonal anti-CD68 (1:200, Immunoway Biotechnology, USA), and rabbit polyclonal anti-NLRP3 (1:200, Boster, China) overnight at 4 ℃, rinsed three times and incubated with secondary antibody. Images were obtained utilizing an ordinary microscope after counterstaining with 3,3’-diaminobenzidine (DAB) staining (ZSGB-BIO, China). The immunohistochemical (IHC) positive staining area was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, USA) to assess average optical density (AOD).36 (link)
6
Histological Analysis of ingWAT
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Collected ingWATs were fixed in 10 % neutral buffered formalin for >24 h. The tissues were then embedded in paraffin, sectioned at 4-5 μm, and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis in ingWAT sections was performed using primary antibodies of fatty acid binding protein 4 (FABP4) (Proteintech, Wuhan, China), KI67 (Proteintech), and uncoupling protein 1 (UCP1) (Proteintech) and visualized with 3, 3′-diaminobenzidine (DAB) staining (ZSGB-Bio, Beijing, China). The slides were examined under a digitalized microscope camera (Olympus, Tokyo, Japan) and further analyzed using the ImageJ software (NIH, Bethesda, MD).
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